Experimental Parasitology 134 (2013) 313–317

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Experimental Parasitology

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Evaluation of ELISA, neck muscle, tongue and eyelid examinations for thediagnosis of swine cysticercosis in a highly endemic area of north India

Aloukick Kumar Singh a, Satyendra Kumar Singh a, Kashi Nath Prasad a,⇑, Amrita Singh a, Anamika Bajpai a,Mohibur Rahman a, Ravi Prakash Rai a, Rakesh Kumar Gupta b, Mukesh Tripathi c, Nuzhat Husain d

a Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, Indiab Department of Radiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, Indiac Department of Anesthesia, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, Indiad Department of Pathology, Dr. Ram Manohar Lohiya Institute of Medical Sciences, Lucknow 226 010, India

h i g h l i g h t s

� Neck muscle, eyelid, and tongue wereexamined for diagnosis of swinecysticercosis.� In house ELISA was developed as

sero-diagnosis tool.� ELISA with crude lysate is a good

diagnostic tool for swinecysticercosis.� Neck muscle, eyelid and tongue

examination can also be used forscreening of swine.

0014-4894/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.exppara.2013.03.033

⇑ Corresponding author. Address: Department ofPostgraduate Institute of Medical Sciences, Lucknow 2Fax: +91 522 2668100/8017.

E-mail address: knprasad@sgpgi.ac.in (K.N. Prasad

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 October 2012Received in revised form 14 March 2013Accepted 20 March 2013Available online 8 April 2013

Keywords:CysticercosisELISANeck muscle examinationCrude lysate

a b s t r a c t

Swine cysticercosis is very common in the developing countries where pigs are raised. Undercooked mea-sly pork consumption leads to taeniasis; Taenia carriers act as source of human and swine cysticercosisand neurocysticercosis. Diagnosis of swine cysticercosis is important to break the cycle of disease trans-mission. The present study compared the neck muscle, tongue and eye examinations, and serum ELISAwith different preparations (crude lysate, cyst fluid, scolex and cyst wall antigens) of Taenia solium cystfor the diagnosis of swine cysticercosis. Total of 24 pigs initially identified by neck muscle, tongue andeyelid examinations were purchased from local slaughter house and subjected to MRI for confirmationof cysticercosis. Sera from 20 MRI confirmed infected pigs and 50 disease free controls were subjectedto ELISA with T. solium cyst antigens. Neck muscle examination was 100% sensitive and 75% specificfor the diagnosis of swine cysticercosis, whereas tongue and eye examinations were 70% and 25% sensi-tive, respectively. ELISA with crude lysate had 85% sensitivity and 98% specificity. ELISA with cyst fluid,scolex and cyst wall antigens showed 70%, 65%, and 45% sensitivity, respectively. The present studyshowed that neck muscle examination was highly sensitive but less specific, while ELISA with crude anti-

ll rights reserved.

Microbiology, Sanjay Gandhi26 014, Uttar Pradesh, India.

).

314 A.K. Singh et al. / Experimental Parasitology 134 (2013) 313–317

gens had reasonable sensitivity and high specificity for diagnosis of swine cysticercosis. ELISA with crudelysate can be used as a screening tool for swine infection.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Taenia solium taeniasis and cysticercosis are still a major publichealth problem in developing countries including India (Prasadet al., 2008; Kanobana et al., 2011; Cordero et al., 2010). The life cy-cle comprises human and pig as intermediate host, harboring larvalcysts (Cysticercus cellulose), whereas human is only the definitivehost harboring adult tapeworm in intestine. When the larva lodgesin the brain, it is called neurocysticercosis (NCC). NCC is identifiedas the leading cause of acquired epilepsy in developing countries(Prasad et al., 2009; Flisser, 2001). Pig breeding has been a familialoccupation of some communities in India and pork is commonlyconsumed in certain communities. The consumption of infectedpork is not only responsible for taeniasis but it also reduces themarket value of pork. Thus T. solium causes economic loss to pigfarmers. In India control measures to prevent human consumptionof infected pork are inadequate. Consumption of infected porkhelps in the persistence of human taeniasis-cysticercosis complexcycle in the endemic areas. Thus there is urgent need to developand introduce control and preventive measures in endemic areaswhere swine are sold on large scale without any inspection. In anearlier study from our center it was found that 26% of the swineslaughtered for human consumption had cysticerci in their skeletalmuscles (Prasad et al., 2002). In a door to door survey in pig farm-ing community of North India, the prevalence of T. solium taeniasiswas 18.6% and active epilepsy 5.8%; NCC was identified in 48% pa-tients with active epilepsy (Prasad et al., 2007, 2009). As publichealth control measure, visual meat inspection is commonly usedfor diagnosis of swine cysticercosis but it is poorly sensitive andit usually detects heavily infected animals only (Boa et al., 2002;Gonzalez et al., 1990). The other procedures commonly used fordiagnosis of swine cysticercosis are tongue and eyelid examina-tions but they also lack sensitivity. Accurate identification of in-fected pigs will help in future control and eradication programs.The need for a diagnostic test over meat inspection and tongueexamination led to the development of serological tests. T. soliumcysticerci are found in almost all tissues of pigs (Boa et al., 2002)but the infected pigs usually remain asymptomatic. A study fromour center identified clinical signs like excessive salivation, blink-ing and tearing in naturally infected swine with NCC (Prasadet al., 2006). Considering the importance of reliable diagnosis ofswine cysticercosis, we compared different diagnostic methods be-tween free roaming naturally infected swine and those reared ingovernment firms. In the present study all the infected pigs wereidentified by neck muscle, tongue and eyelid examinations andconfirmed by magnetic resonance imaging (MRI). Sera from pigswere used to detect antibody by ELISA with different parasite prep-arations such as crude lysate, cyst fluid, cyst wall and scolexantigen.

2. Materials and methods

2.1. Study subjects

A total of 24 animals were initially identified and selected onthe basis of clinical signs as described earlier (Prasad et al.,2006). All these animals underwent neck muscle, tongue and eye-lid examinations for the presence of cysticerci. All the animalswere also subjected to MRI of the head and neck region to detectcysticerci in head and neck muscles, tongue and brain. Sera from

all the MRI confirmed animals and 50 sera samples from pigs thatwere not allowed to roam freely and were reared in governmentfirms were tested for antibody by in-house ELISA with differentparasitic preparations. This work was approved by institute’s eth-ical committee.

2.2. Tongue examination

The tongue was examined for the presence of cysticerci by lin-gual palpation method as described earlier for the presence of cystlike nodule (Githigia et al., 2002; Ngowi et al., 2004; Phiri et al.,2003). Briefly a hard wooden rod or cotton rope is used to keepthe mouth open and the tongue was gently pulled out andexamined.

2.3. Neck muscle examination

Skeletal muscle of the neck was examined for the detection ofcysticerci. A small skin incision 4–5 cm long was made on the lat-eral side of the neck and the muscle was examined by inserting fin-ger by trained volunteers to palpate nodule, if any.

2.4. Eyelid examination

The eyelids of all the swine were examined for the presence ofcysticercus cyst nodule by direct visualization.

2.5. MRI

MRI was performed according to the protocol already devel-oped by our center (Prasad et al., 2006). During MRI the blood fromeach swine was collected from ear vein, serum was separated andstored at �80 �C for further use.

2.6. Preparation of T. solium cyst antigen with different fragments

Antigen from different fragments of cyst was prepared as de-scribed in our earlier study (Prasad et al., 2011). Briefly the anti-gens were prepared as described below.

2.6.1. Whole crude lysateThe crude lysate (CL) of the cyst was prepared following the

standard protocol. In brief, the cysts were isolated from the mus-cles of the pig and washed five times in phosphate buffered saline(PBS) with protease inhibitor cocktail (Sigma, USA), homogenizedand centrifuged. The soluble antigens in the supernatant were col-lected, filtered through 0.45 lm filters and stored at �80 �C till fur-ther use.

2.6.2. Cyst fluidCyst fluid (CF) was aspirated from the freshly isolated pig mus-

cle T. solium cysts. The fluid was sonicated at 20 kHz for 4 min with1 min pulse each, and then centrifuged at 200,000g for 2 h andsupernatant was collected. Protease inhibitor cocktail (SigmaUSA) was added to the supernatant and stored at �80 �C till furtheruse.

2.6.3. Cyst wallThe residuals left after aspirating the fluid from T. solium cysts

were used as the cyst wall (CW) antigen. The residuals were

Fig. 2. Cyst like nodule (arrows) on tongue of swine.

Fig. 3. Presence of cysticerci in eyelids of swine.

A.K. Singh et al. / Experimental Parasitology 134 (2013) 313–317 315

washed three times with PBS, homogenized vigorously under cool-ing condition and then centrifuged at 200,000g for 2 h. Supernatantwas collected and protease inhibitor cocktail was added.

2.6.4. ScolexAfter the collection of cyst fluid and cyst wall, the scolices were

collected, washed three times with PBS, homogenized under cool-ing condition and then centrifuged at 200,000g for 2 h. Supernatantwas collected and protease cocktail inhibitor was added.

Resultant proteins were quantified by Lowry’s method.

2.7. Elisa

ELISA was performed with different parasitic preparations forthe detection of IgG antibodies in sera of cysticercosis positiveand control animals. A checker board titration was performed toknow the best concentration of antigen for coating the microplatesand the best dilution of sera and conjugate. For microplate coating,antigen preparations were diluted in 0.05 M carbonate-bicarbon-ate buffer pH 9.6. Maxisorpmicroplates (Nunc, Denmark) werecoated with 100 ll of diluted antigen, incubated at 37 �C for 1 hand then for 12 h at 4 �C. After end of each steps of the reactionmicroplates were washed three times for 5 min with PBS-T(0.01 M phosphate-buffer saline, pH 7.2 containing 0.05% Tween20). Blocking was done by incubation with 10% skim milk in0.05 M carbonate-bicarbonate buffer 200 ll/well. Test sera andconjugate (Koma biotech, Korea) were diluted in PBS-T containing5% skim milk and incubated for 1 h at 37 �C. Then substrate wasadded and after 5 min the reaction was stopped with 50 ll of 2NH2SO4. Finally the optical density was taken at 492 nm using anELISA reader (Inverness Microplate reader, India).

2.8. Statistical analysis

Data were analyzed with SPSS statistical software, version 16.0(SPSS Inc., Chicago, IL, USA). Receiver operating characteristic(ROC) curve was constructed to evaluate the cutoff value of ELISAthat best differentiated controls from those with cysticercosis.

3. Results

Among the 24 animals that were selected on the basis of clinicalsigns, presence of cysticerci in muscles was detected in 20 animalswith brain involvement in 18 by MRI (Fig. 1). Twenty-one animalswere positive by neck muscle, 14 by tongue (Fig. 2) and 05 by eye-lid examinations (Fig. 3) (Table 1). Sera from 20 MRI confirmed and50 healthy animals from commercial firms were subjected to ELISAwith different antigens. The sensitivity and specificity of different

Fig. 1. In vivo axial oblique MR image. (A) shows cysts (arrows) within brain andneck muscle region and (B) shows cysts on tongue.

methods for the diagnosis of cysticercosis were determined usingMRI as gold standard.

3.1. Sensitivity and specificity of different fragments of T. solium cyst

After standardization by checkerboard titration, ELISAs wereperformed using the antigen concentrations (crude lysate, cystfluid, scolex and cyst wall) at 5 lg/ml, with serum diluted at1:4000 and conjugate at 1:800. Based on optical density (OD) re-sults from healthy animals, a cut-off OD value was determinedusing receiver operating characteristics (ROC) curve. The area un-der curve (AUC) was 0.982, 0.980, 0.907, and 0.854 for crude lysate,cyst fluid, scolex and cyst wall antigen respectively (Fig. 4). The cutoff value was 0.322 for crude lysate, 0.402 for cyst fluid, 0.257 forscolex and 0.404 for cyst wall antigen. Sera with absorbance higherthan this cut-off value at 492 nm were considered positive. Thesensitivity and specificity of all four preparations were compared.The crude lysate, cyst fluid, scolex and cyst wall had sensitivity85%, 70%, 65%, and 45%, and specificity of 98%, 98%, 96%, and 98%respectively for the diagnosis of swine cysticercosis (Table 2).

3.2. Sensitivity and specificity of neck muscle, tongue and eyelidexamination

Neck muscle, tongue and eyelids were thoroughly examined forthe presence of cysticerci. Neck muscle examination was positivein 87.5% animals whereas tongue and eyelid examination showed58.33% and 20.83% positivity. Neck muscle examination was found

Table 1Comparison of neck muscle, tongue and eyelid examinations with magnetic resonance imaging (MRI) as gold standard.

Methods MRI (Gold Standard) Sensitivity

MRI +ve MRI �ve Total Specificity (%)

Neck muscle examination +ve 20 01 21 100Neck muscle examination �ve 00 03 03 75Total 20 04 24

Tongue examination +ve 14 00 14 70Tongue examination �ve 06 04 10 100Total 20 04 24

Eyelid examination +ve 05 00 05 25Eyelid examination �ve 15 04 19 100Total 20 04 24

Fig. 4. Shows ROC curve of crude lysate. (A) AUC was 0.982, cyst fluid. (B) AUC was 0.980, scolex antigen. (C) AUC was 0.907 and cyst wall antigen and (D) AUC was 0.854,respectively.

Table 2Comparative efficacies of different parasitic antigens by ELISA.

Crude lysate Cyst fluid Scolex Cyst wall

Sensitivity 85% (17/20) 70% (14/20) 65% (13/20) 45% (9/20)Specificity 98% (49/50) 98% (49/50) 96% (48/50) 98% (49/50)

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to have higher sensitivity and specificity (100% and 75%), followedby tongue examination (70% and 100%) and eyelid examination(25% and 100%). Table 1.

4. Discussion

To the best of our knowledge, this is the first study where theresults of ELISA, neck muscle, tongue and eyelid examinations

were compared with MRI for the diagnosis of swine cysticercosis.Demonstration of scolex by MRI has been described as a definitivecriterion for diagnosis of NCC in human (Del Brutto et al., 2001). Inthe present study scolex could be demonstrated in all the infectedpigs; hence MRI was used as the reference standard keeping inmind doing histopathology on live animal will not be possible ina real pig farm setting. We performed ELISA with four differentpreparations of T. solium cyst antigen. The best results were ob-tained with crude lysate having sensitivity of 85% and specificityof 98%. Several other studies also reported similar results with sen-sitivity ranging between 70–88% (Gonzalez et al., 1990; Pathaket al., 1994; Pinto et al., 2000; Nunes et al., 2000). ELISA performedwith cyst fluid antigen showed 70% sensitivity and 98% specificityand our results were inconsistent with an earlier study in naturallyinfected swine where ELISA using this antigen showed sensitivity

A.K. Singh et al. / Experimental Parasitology 134 (2013) 313–317 317

of 55.5%; however, the sensitivity was higher (86%) using the sameantigen in experimentally infected pigs (Scuitoo et al., 1998). Thepossible reason for higher sensitivity in our study was that almostall pigs included were heavily infected. Our results are in agree-ment with findings of Nunes et al. (2000) who reported sensitivityof 70%. In an Indian study higher sensitivity and specificity (91%and 92%, respectively) was reported with scolex antigens (Kumarand Gaur, 1987). But we observed lower sensitivity with scolexantigen (65%). ELISA with cyst wall antigen had the lowest sensitiv-ity (45%). Although the specificity was higher (98%), due to lowsensitivity, cyst wall based ELISA may not be suitable for the diag-nosis of cysticercosis. Since this antigen had not been used in swinecysticercosis, our data cannot be compared. However, sera frompatients with proven neurocysticercosis cases were least reactiveusing the cyst wall antigen (Baily et al., 1988). Thus the resultsshowed that ELISA using crude antigens had better sensitivityand specificity compared to scolex, cyst wall and cyst fluid anti-gens for the diagnosis of swine cysticercosis.

Neck muscle examination was found 100% sensitive for thediagnosis of swine cysticercosis. Thus it is evident from our resultsthat neck muscles are the most common site of infection by cysti-cerci. Similar findings were reported from Peru (Gonzalez et al.,1990). Moreover, 90% of neck muscle positive animals also hadbrain involvement, thus neck muscle examination may be helpfulin identification of swine NCC where imaging facilities are lackingand these swine can be used as an experimental model of NCC.

The utility of the tongue examination in detecting porcine cys-ticercosis had previously been questioned (Boa et al., 2002; Phiriet al., 2002). This method requires technical expertise, is of lowsensitivity, and is capable of detecting C. cellulose cysts only inheavily infected pigs with sensitivity of 70% and specificity of100%. The sensitivity of tongue examination varied from 43% to70% in most endemic areas elsewhere in the world (Gonzalezet al., 1990; Phiri et al., 2002). However, eyelid examination wasonly 25% sensitive and 100% specific. A study from Iranian Jaya,Indonesia, four of the six infected swine had eyelid involvementand only one had tongue involvement (Subahar et al., 2001). Thisdiscrepancy might be due to the small number of heavily infectedpigs included in that study.

Finally we conclude that neck muscle examination and ELISAwith crude antigen may be useful for the diagnosis of swine cysti-cercosis in endemic regions. We also suggest that one should ini-tially screen the swine on the basis of clinical signs as reportedearlier (11), then examine eyelids for the presence of ocular cystfollowed by tongue and neck muscle examination. These all exam-inations are not only cost effective but also of less time consumingand only required trained workers.

Financial support

The study was supported by the Department of Science andTechnology, Government of India through Grant No. SR/SO/HS-21/2008.

Acknowledgments

The authors Mr S.K. Singh (09/590/(0147)/2010-EMR-I)acknowledge the Council of Scientific and Industrial Research andMs. Anamika Bajpai (Ref. No. 2040) University Grant Commission,Government of India, New Delhi.

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